Project Summary Aging is associated with cognitive decline, neurodegeneration, and brain atrophy, and is also the most important risk factor for sporadic Alzheimer's disease (AD). Compelling evidence links mitochondria biology to age-associated disease vulnerability, including AD. Mitochondria are highly dynamic organelles that adapt to metabolic changes including metabolite availability. Our recent discoveries, using entirely novel mouse models, have prompted us to interrogate acetyl-CoA directed mitochondrial adaption and its role in aging and AD pathogenesis. N?-lysine acetylation uses acetyl-CoA as the acetyl donor and was initially thought to occur only in the cytoplasm, mitochondria, and nucleus; however, in 2007 we discovered that the endoplasmic reticulum (ER) is also able to acetylate newly-synthesized polypeptides. Since then, we have successfully identified the entire biochemical machinery responsible for ER-acetylation and generated relevant animal models. The machinery includes AT-1/SLC33A1, which translocates acetyl-CoA from the cytosol to the ER lumen. In studying AT-1 relevant mouse models, we have discovered that the cytosol-ER acetyl-CoA pathway may directly contribute to aging and AD: systemic overexpression of AT-1 in the mouse causes a progeria-like phenotype, and conversely, genetic and biochemical inhibition of ER acetylation rescues AD neuropathology. Furthermore, changes in cytosol-ER flux induce mitochondrial and epigenetic changes. The GENERAL HYPOTHESIS of this research is that the cytosol-to-ER flux of acetyl-CoA maintains mitochondrial homeostasis through epigenetic regulation of gene expression. Alteration of this balance can influence the progression of aging and AD. This grant targets the cytosol-ER cross-talk to uncover mechanisms that regulate (i) how mitochondria adapt to changes in acetyl-CoA flux within the cell and (ii) how these changes affect the brain as a function of aging and AD. Specific Aim 1 will determine the impact of brain aging and AD neuropathology on mitochondrial biochemistry and metabolism. This Aim will use high-resolution proteomics and will target (i) normally aged mouse and primates; (ii) mice with accelerated and delayed aging; (iii) AD brain tissue; and (iv) iPSC-derived human neurons. Specific Aim 2 will dissect the molecular mechanism(s) responsible for the mitochondria adaptation that occurs in the brain as a function of aging and AD. It will target mitochondria bioenergetics and TCA pathway engagement, mitochondria dynamics, and the transcriptional machinery responsible for acetyl-CoA flux-directed mitochondrial adaptation. Specific Aim 3 will establish the cytosol-to-ER flux of acetyl-CoA as a therapeutic target in accelerated and normative aging. We have identified a novel compound that disrupts the above flux and rescues AD neuropathology in the mouse. We will test the ability of this compound to rescue cognition, neurodegeneration, and mitochondrial adaptation in AT-1 induced progeria and in normal aging. The proposed research has strong translational potential relevant to both aging and AD.